RESEARCH ARTICLE
Guanine-Based Structural Coloration as an Indicator of Oxidative Stress in a Cichlid Fish MATTHEW D. CAHN1, ALEXANDRIA C. BROWN1,2, 1 AND ETHAN D. CLOTFELTER * 1
Department of Biology, Amherst College, Amherst, Massachusetts Graduate Program in Organismic and Evolutionary Biology, University of Massachusetts, Amherst, Massachusetts 2
ABSTRACT
J. Exp. Zool. 323A:359–367, 2015
Vertebrate pigmentation is known to be influenced by oxidative stress, but few studies have tested the hypothesis that structural coloration can be similarly affected. We tested whether fish iridophores, which produce structural color using guanine stacks, might be affected by the prooxidant–antioxidant balance of the animal. Specifically, we hypothesized that convict cichlids (Amatitlania nigrofasciata) metabolize guanine present in iridophores to uric acid, an antioxidant, in response to oxidative damage. We used Hunter's contrast gloss and high performance liquid chromatography to determine whether dietary guanine supplementation allows fish to maintain their structural coloration despite oxidative stress induced via ultraviolet-B (UV-B) radiation. We found that dietary guanine was associated with greater skin gloss, and that exposure to UV-B light reduced glossiness. UV-B exposure did not increase oxidative damage (acrolein) or total antioxidant capacity in the skin or liver. Our experiment did not detect effects of dietary guanine or UV-B light on uric acid, but uric acid was positively related to antioxidant capacity. Our results support the hypothesis that structural color in fish may be altered by environmental stressors such as exposure to UV light, and highlight the need for future studies to consider the role of iridophores in condition-dependent visual signaling. J. Exp. Zool. 323A:359–367, 2015. © 2015 Wiley Periodicals, Inc. How to cite this article: Cahn MD, Brown AC, Clotfelter ED. 2015. Guanine-based structural coloration as an indicator of oxidative stress in a cichlid fish. J. Exp. Zool. 323A:359–367.
A large body of literature suggests that pigment-based signals can indicate the condition of the signaler in a variety of contexts: immune condition (Blount et al., 2003; McGraw and Ardia, 2003), nutritional condition (Hill and Montgomerie, '94), and oxidative stress (Pike et al., 2007). However, animal coloration can also be produced by structural elements. Structural coloration is an endogenous mechanism for producing color. In birds, structural coloration arises from combinations of keratin, melanin, and air (Doucet et al., 2006). The striking varieties of colors expressed on butterfly wings are also produced by structural color (Saranathan et al., 2010). In both taxa, previous studies suggest that structural coloration can act as an important signal underlying male and
Grant sponsor: National Science Foundation, Division of Integrative Organismal Systems; grant number: #1051598; grant sponsor: Dayton Fund at Amherst College, the Department of Biology at Amherst College. Alexandria C. Brown’s current address: Division of Biostatistics and Epidemiology, School of Public Health and Health Sciences, University of Massachusetts, Amherst, MA. Correspondence to: Ethan D. Clotfelter, Department of Biology, AC#2237, Amherst College, Amherst, MA 01002. E-mail: [email protected] Received 25 July 2014; Revised 30 January 2015; Accepted 2 February 2015 DOI: 10.1002/jez.1926 Published online 6 April 2015 in Wiley Online Library (wileyonlinelibrary.com).
© 2015 WILEY PERIODICALS, INC.
360 female mate preferences (e.g., nymphalid butterflies Hypolimnas bolina, Kemp, 2007; blue tits Cyanistes caeruleus, Hunt et al., '99; Eastern bluebirds Sialia sialis, Siefferman and Hill, 2005). In contrast to the species mentioned above, structural coloration in fish (Levy-Lior et al., 2010), cephalopods (M€athger et al., 2009), reptiles (Cooper and Greenberg, '92), spiders (LevyLior et al., 2010; Hsiung et al., 2014), and amphibians (Taylor, '69) consists of specialized cells containing alternating layers of cytoplasm and guanine platelets. It is thought that highly refractive guanine crystals and less refractive cytoplasm alternate in multilayer stacks within the iridophores to refract light, creating its optical characteristics from the interference of light reflected from the layer (Ide and Hama, '72; Rohrlich and Rubin, '75; Levy-Lior et al., 2010). In fishes, structural coloration is responsible for the metallic luster, which may provide camouflage from multiple angles due to light scattering produced by these guanine platelets (McKenzie et al., '95) and may play a role in the production of some color patterns (e.g., zebrafish Danio rerio, Hirata et al., 2003). Although guanine can be synthesized endogenously, guanine-based iridophore expression has been shown to depend upon condition in lizards (Brown et al., '48; San-Jose et al., 2013). Furthermore, studies on economically important fishes have shown that dietary supplementation with guanine can enhance immunity and growth (Ramadan et al., '94; Li et al., 2004a,b). These results suggest that guanine-based structural coloration may be costly for the organism to create or maintain. The current study tests whether the light-scattering ability of iridophores is linked to physiological condition, specifically the prooxidant–antioxidant balance. Free radicals, also known as reactive oxidative metabolites (ROMs), are a family of highly reactive molecules that can damage intracellular components (Dowling and Simmons, 2009). Elevated levels of free radicals have been linked to reduced expression of other conditiondependent traits, such as pigment-based color (von Schantz et al., 1999), and may play a role in aging (Balaban et al., 2005), immune function (Chew and Park, 2004), and life history traits (Blount et al., 2003; Pike et al., 2007). When an organism cannot counter the effects of free-radical production, oxidative stress occurs (Monaghan et al., 2009). Uric acid is an efficient scavenger of singlet oxygen, hydroxyl radicals, and peroxyl radicals (Sautin and Johnson, 2008), and has been shown previously to act as an antioxidant in fishes (Xue et al., '98; Ciereszko et al., '99). Oxidative stress may affect iridophore-based structural coloration via a physiological tradeoff: guanine allocation to iridophores could decrease circulating levels of uric acid, a metabolite of guanine and an antioxidant, available for mediating detrimental effects of free radicals. Condition dependence could be enforced by the energetic or opportunity cost of synthesizing guanine molecules and assembling the molecules in the precise manner necessary for J. Exp. Zool.
CAHN ET AL. iridophore function, as suggested previously by other researchers (Li and Gatlin, 2006; Levy-Lior et al., 2008). In our study, we used Hunter's contrast gloss to quantify iridophore expression in a fish. Hunter's contrast gloss has been used to measure luster in various industrial applications (Nickerson, '57; O’Brien et al., 1984). More recently, Hunter's contrast gloss has been used to quantify the luster of birds’ feathers (Maia et al., 2011) and eggshells (Igic et al., 2015). This measure takes advantage of the physical properties of gloss or luster; if a larger proportion of light is reflected at an angle equal to the incident angle (specular reflectance) relative to light reflected at random angles (diffuse reflectance), the surface will appear glossier. Hunter's contrast gloss can be calculated as the ratio of specular to diffuse reflectance, as shown below: Hunter’s Contrast Gloss ¼
700nm
S
l¼350nm
Specular ReflectanceðlÞ Diffuse ReflectanceðlÞ
We validated Hunter's contrast gloss as a measure of guanine in fish skin using high performance liquid chromatography (HPLC). We hypothesized that elevated free radical levels would increase guanine conversion to the antioxidant uric acid, resulting in reduced structural color. Dietary guanine supplementation may prevent this reduction in structural coloration if guanine is limited by access to the carbon and nitrogen building blocks needed to assemble it. We tested these predictions using the convict cichlid (Amatitlania nigrofasciata). Convict cichlids are often used in behavioral studies (Wisenden, '95; Beeching et al., '98) and females possess ornamental coloration that males lack (Wisenden, '94). Several studies have investigated the pigmentation component of convict cichlid coloration (Beeching et al., 2002; Anderson et al., 2014; Brown et al., 2014), but, to date, the concept of gloss has not been used in this or any other species of fish to quantify structural coloration. To test the hypothesis that iridophore-based expression can be altered by oxidative stress in convict cichlids, a 2 2 experiment design was used in which fish were fed a diet supplemented with guanine or a non-supplemented control diet. Across these diet groups, fish were then exposed to ultraviolet-B (UV-B) light at a level shown previously to induce a stress response in fish (Salo et al., 2000), or to control lighting conditions. Spectral reflectance of the skin, quantities of uric acid and guanine in the skin, and oxidative stress parameters were measured to determine the effects of diet and UV-B light on gloss. If increased free radical production reduces gloss, this result will support the hypothesis that iridophore appearance may be related to condition. If this reduction in gloss is accompanied by an increase in uric acid in the skin, fish may be metabolizing the guanine in their iridophores to combat oxidative stress. Finally, if these negative effects can be rescued by the addition of guanine to the diet, iridophore-based color expression may also contain information about a signaler's
STRUCTURAL COLORATION AND OXIDATIVE STRESS environment and ability to absorb, transport, and build necessary molecules.
MATERIALS AND METHODS Study Animals and Experimental Diets Sexually mature female convict cichlids (A. nigrofasciata), which we identified by the presence of orange ventral coloration, were obtained from a commercial distributor. Fish acclimated to the laboratory for 2 days prior to being housed in individual 7-L tanks at 26 °C. Water was aerated with air stones and changed (50–75%) twice weekly. All handling and treatment of animals was approved by the Institutional Animal Care and Use Committee (IACUC) of Amherst College. The diet was a modified H440 diet with a low-nutrient base of cellulose, dextrin, and agar (Ellis et al., 2000; Brown et al., 2014). Pure guanine (no. 190691 MP Biomedicals, Santa Ana, CA, USA) was added to the supplemented diet at a concentration of 4 mg g1, a concentration consistent with other dietary nucleotide supplementation studies (Ramadan et al., '94; Li et al., 2004a,b). Fish were fed to satiation once daily with the guanine-supplemented diet or the nonsupplemented diet for 5 weeks prior to the oxidative challenge. Ultraviolet Light Exposure After 5 weeks on the experimental diets, half of the fish from each diet treatment group were exposed to UV-B radiation, while the other half was exposed to control lighting conditions. Fish were removed from their home tanks and placed in identical 7-L tanks placed 20 cm under a pair of Philips TL 40 W/12 RS light bulbs, which each emitted 40 W of UV-B light from 290–315 nm (Philips, Amsterdam, Netherlands). The UV-B light exposure lasted for 120 min, a dosage previously found to be non-lethal yet elicit stress responses in another fish, the common roach Rutilus rutilus (Salo et al., 2000). After irradiation, fish were returned to their original tanks and allowed to recover for 24 hr. Control fish were placed in the same tanks as the UV-B exposed fish for 120 min, but with the UV-B bulbs turned off.
361 Morphological and Color Measurements Fish were anesthetized 24 hr after UV-B exposure with a 2 g L1 tricaine methanesulfonate (Western Chemical Co., Ferndale, WA, USA) solution. Standard length (0.01 mm) and body mass (0.01 g) were measured prior to structural coloration quantification using spectrometry. Fish were placed on a damp sponge and spectrometric readings were taken 2 mm posterior from the operculum on each fish (Fig. 1A and B), a planar location typically lacking pigmentation. A deuterium-tungsten halogen lamp fiber-optic light source (Ocean Optics Jaz1, Dunedin, FL, USA) was mounted 5 mm above and 45° from the area to be measured. Opposite the light source, an Ocean Optics USB4000 spectrometer probe 5 mm from the fish collected and measured the light reflected at 45° and then at 25° (Fig. 1A). Reflectance spectra from the spectrometer were recorded using SpectraSuite software (version 2.0.159). Spectra were analyzed between 350 and 700 nm (Jackson, 2003; Brown et al., 2014). The spectrometer was set to use boxcar smoothing of five with three spectra averaged with an integration time of 100 ms. The ratio of specular reflectance (light captured by the detector at 45°) to diffuse reflectance (light captured by the detector at 25°) was used to quantify Hunter's contrast gloss (see equation above; Hunter, '37; Rasmussen and Dyck, 2000; Maia et al., 2011). We found that the amount of guanine in the skin (determined by high performance liquid chromatography, see below) significantly predicted the gloss measurement in the same region of the body (R2 ¼ 0.14, F1,35 ¼ 5.24, P ¼ 0.03; Fig. 2). Thus, we will hereafter consider gloss to be an indicator of the structural coloration in the skin. For all subsequent analyses involving gloss, the change in gloss from immediately prior to 1 day after UV-B exposure (or control) was used. Positive values indicate an increase, and negative values a decrease, in glossiness. After morphological and color measurements, fish were euthanized by cervical dislocation. Liver and skin samples were aseptically removed with surgical scissors and forceps and stored at 80 °C for later analysis.
Figure 1. Apparatus used to obtain Hunter's contrast gloss measurements from convict cichlids A. nigrofasciata, showing the location of the spectrometer probe and light source (A) and lateral view of a fish, showing the location of spectrometer measurements taken on live fish (B). Gloss measurements were taken 2 mm posterior of operculum, a location lacking melanin pigmentation. J. Exp. Zool.
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Figure 2. Guanine content in the skin (as measured by high performance liquid chromatography) and Hunter's contrast gloss of the skin of female A. nigrofasciata were positively correlated (R2 ¼ 0.14, F1,35 ¼ 5.24, P ¼ 0.03). The gloss measurement was sine transformed due to skew.
Uric Acid and Guanine Measurements To measure uric acid in the liver, livers were homogenized in 1 mL mg1 phosphate buffered saline and centrifuged. The uric acid content of the supernatant was determined by using a colorimetric uric acid assay kit (QuantiChrom no. DIUA-250, BioAssay, Hayward, CA, USA) prepared according to the manufacturer's instructions. Liver uric acid was quantified using a microplate reader (Bio-Tek, Winooski, VT, USA) at 590 nm. High performance liquid chromatography (HPLC) was used to quantify guanine and uric acid from the skin samples using a protocol adapted from Burdett et al. (2012). In brief, skin samples were homogenized in 20 volume (v:w) 0.52 mM sodium 1-pentanesulfonate (no. 76952 Sigma–Aldrich, St. Louis, MO, USA) and 0.20 M KH2PO4 (no. P5655 Sigma–Aldrich) monobasic at pH 3.5 mobile phase. Samples were centrifuged at 16,000g for 15 min and the supernatant was filtered through 0.22 mm Spin-X cellulose acetate filter tubes (Sigma–Aldrich) at 16,000g for 5 min. The filtrate was run on a Waters 600 HPLC (Waters Corp., Milford, MA, USA) using a C18 column. The flow rate was set at 1.0 mL min1 with an injection loop size of 12 mL. AWaters 600 E wavelength absorbance detector produced a chromatogram at 254 nm. Peaks were integrated and compared to a guanine standard by the Empower 3 HPLC software (Waters Corp.). Oxidative Stress Assays Two assays were used to measure oxidative stress parameters in the fish. The first, which measures acrolein, detects lipid J. Exp. Zool.
peroxidation (Uchida et al., '98; Kurtz et al., 2006). The second assay was a Trolox equivalent antioxidant capacity (TEAC) assay, which measures the ability of a biological sample to quench free radicals as compared to Trolox, a synthetic analog of vitamin E. For the acrolein assay, liver protein was adjusted to 1 mg mL1 using a Bradford protein assay (Bio-Rad, Hercules, CA, USA). As described previously (Kurtz et al., 2006; Brown et al., 2014), the acrolein assay was performed using the monoclonal antibody (mAb5F6, no. ADI-KAMCC101-E, Enzo Life Sciences, Farmingdale, NY, USA) and horseradish peroxidase antibody from mice as the secondary antibody (no. A9044 Sigma–Aldrich). SigmaFast OPD (no. P9187) was the probe that allowed for acrolein quantification at 474 nm. Standards were compared to a standard curve of acrolein-modified bovine serum. Amounts of acrolein are expressed as mM acrolein mg1 total protein. The TEAC assay quantified the antioxidant capacity of fish livers and skin relative to Trolox (Amar et al., 2000; Raghavan et al., 2008). Liver and skin homogenates were prepared as for the acrolein assay. The assay uses 2,20 -azino(3-ethylbenzthiazoline-6-sulphonic acid) (no. A1888 Sigma–Aldrich), which has a blue color in its free-radical form and is colorless when quenched. Thus, lower TEAC values due to reduced absorbance in the spectrometer are indicative of greater antioxidant capacity. Serial dilutions of Trolox were used to construct the standard curve. The antioxidant capacities of Trolox and the
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homogenates were quantified at 734 nm using a microplate reader (Bio-Tek, Winooski, VT, USA). Statistical Analyses All statistical tests were performed using R version 3.0.1. Log transformations (liver TEAC) or sine transformations (liver acrolein, skin TEAC, and Hunter's contrast gloss) were applied to achieve normality of residuals. The effect of dietary guanine supplementation and UV-B exposure on structural coloration and oxidative stress parameters was compared using ANCOVA. Linear regression tested the relationship between structural coloration and oxidative stress parameters or guanine content of the skin, with body condition as a covariate. Linear regression also tested the effect of uric acid on oxidative stress. Fulton's body condition (Bolger and Connolly, '89; Meka and McCormick, 2005; Robinson et al., 2008) was calculated using the following equation: body mass (standard length3)1, and was included in analyses where it was significant. Differences were considered statistically significant when P < 0.05.
RESULTS Data were collected from 66 female A. nigrofasciata, which had a mean ( s.e.m.) body mass of 1.89 0.47 g and a mean standard length of 3.77 0.47 mm. Thirteen fish were assigned to the control diet þ no UV-B group, 14 fish were assigned to the control diet þ UV-B group, 15 fish were assigned to the guaninesupplemented diet þ no UV-B group, and 13 fish were assigned to the guanine-supplemented diet þ UV-B group. In analyses with n < 66, differences in sample sizes were due to fish tissues or organs too small to analyze. The reflectance spectra obtained from a representative fish are shown in Figure 3; these values were used to calculate our gloss measurement. We found no differences in skin gloss between convict cichlids maintained on the guanine diet compared to the control diet (F1,45 ¼ 0.80, P ¼ 0.38; Fig. 4), but as described above, the guanine content of a fish's skin was positively related to its gloss (R2 ¼ 0.14, F1,35 ¼ 5.24, P ¼ 0.03; Fig. 2). Skin gloss was significantly reduced by exposure to UV-B light (F1,45 ¼ 5.47, P ¼ 0.02; Fig. 4). The interaction between guanine diet and UV-B light was not significant (F1,45 ¼ 1.26, P ¼ 0.27). We quantified acrolein in the liver as an indicator of oxidative damage, and used the Trolox equivalent antioxidant capacity (TEAC) assay to measure the antioxidant capacity of the liver and the skin. To verify these measures, we regressed liver acrolein against liver TEAC and found (as predicted) a significant negative relationship (R2 ¼ 0.27, F1,40 ¼ 15.9, P ¼ 0.0003). When we exposed convict cichlids to UV-B light for 2 hr and collected tissue samples the following day, there were no significant changes in liver acrolein (F1,47 ¼1.55, P ¼ 0.22), liver TEAC (F1,38 ¼ 0.80, P ¼ 0.38), or skin TEAC (F1,46 ¼ 0.03, P ¼ 0.87). Furthermore, exposure to UV-B light did not appear to affect
Figure 3. Reflectance spectra (from 350–700 nm) of a representative A. nigrofasciata. The solid line shows the specular reflectance with both the spectrometer probe and light source at 45°. The dashed line shows the diffuse reflectance with the light source at 45° and the spectrometer probe at 25°. These values were used to calculate our gloss measurement (see Materials and Methods).
the conversion of guanine to uric acid, as uric acid levels were similar between the UV-B and control light groups (F1,47 ¼ 0.01, P ¼ 0.92). Fish fed the diet supplemented with guanine showed no significant differences in liver acrolein (F1,47 ¼ 0.11, P ¼ 0.74), liver TEAC (F1,38 ¼ 0.18, P ¼ 0.67), or TEAC in the skin (F1,46 ¼ 0.02, P ¼ 0.88). There were no significant differences in the amount of uric acid in the liver between fish on the guanine diet and fish on the control diet (F1,47 ¼ 0.53, P ¼ 0.47). The change in skin gloss during the experiment was not a significant predictor of acrolein in the liver (R2 ¼ 0.004, F1,43 ¼ 0.16, P ¼ 0.69), the antioxidant capacity (TEAC) of the liver (R2 ¼ 0.005, F1,39 ¼ 0.19, P ¼ 0.67), or the antioxidant capacity of the skin (R2 ¼ 0.006, F1,42 ¼ 0.24, P ¼ 0.63). Furthermore, the change in glossiness did not predict the amount of uric acid present in the liver (R2 ¼ 0.017, F1,43 ¼ 0.78, P ¼ 0.38). Uric acid was not a significant predictor of acrolein in the liver (R2 < 0.001, F1,49 < 0.001, P ¼ 0.99), but it did explain variation in antioxidant capacity of the liver (P ¼ 0.03; Table 1). Uric acid was not a significant predictor of the antioxidant capacity of the skin (R2 < 0.08, F3,46 ¼ 1.35, P ¼ 0.27). J. Exp. Zool.
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Figure 4. Change in skin gloss following UV-B exposure in female A. nigrofasciata (mean s.e.m.) maintained on a control diet or a guanine-supplemented diet. The symbols represent fish exposed to UV-B light (triangles) and control light conditions (circles). The gloss measurement was sine transformed due to skew. UV-B exposure significantly reduced skin gloss (F1,45 ¼ 5.47, P ¼ 0.02). Neither diet (F1,45 ¼ 0.80, P ¼ 0.38) nor the diet UV interaction (F1,45 ¼ 1.26, P ¼ 0.27) had an effect on gloss.
DISCUSSION In this study, we tested the hypothesis that structural coloration in female convict cichlids A. nigrofasciata is an indicator of physiological condition. We used gloss as a proxy for structural color and then measured changes in gloss in fish fed a diet supplemented with guanine (precursor to the antioxidant uric acid) and in fish in which we disrupted the prooxidant– antioxidant balance via exposure to UV-B light. The first prediction was that glossier skin is associated with greater levels of guanine in the skin, a prediction for which we found some support. We used high performance liquid chromatography (HPLC) to quantify the guanine in fish skin, and found that it was positively related to the gloss measurement taken at that same location when all fish in the experiment were pooled together. However, when we experimentally manipulated fish
diets, we found no differences in skin gloss between diet treatment groups. This absence of an effect may be due to several factors: modest sample sizes; insufficient duration of the diet period (Brown et al., 2014); individual variation in food intake rates (Martins et al., 2011); individual variation in the rates at which guanine is assimilated and deposited in the skin; and competing needs for guanine for other physiological functions. However, it is also possible, contrary to our hypothesis, that dietary guanine supplementation does not affect guanine deposition or skin gloss. Our second prediction was that structural coloration in female convict cichlids is an indicator of oxidative stress. Again, our findings provide mixed support for this prediction. Exposure to UV-B light caused a significant reduction in skin gloss in our experiment. However, UV-B exposure caused no measurable increases in liver acrolein, a lipid peroxidation product widely used as biomarker for oxidative stress (Uchida et al. '98), nor did it decrease antioxidant capacity in either the liver or the skin. Previous studies have shown that UV radiation causes oxidative stress in marine environments (Palenik et al., '91; Dahms and Lee, 2010). Therefore, our lack of effect could be caused by an inability to detect a change in oxidative stress, perhaps due to insufficient UV-B exposure. Nevertheless, UV radiation has also been shown to alter DNA directly, depress immune function, and induce apoptosis in fishes (Ahmed and Setlow, '93; Blazer et al., '97; Salo et al., 2000). Therefore, if UV-B exposure did not induce detectable levels of oxidative stress, differences between UV-B exposed and control groups in terms of iridophore expression can be attributed to its role as a stressor of various physiologically relevant activities. The non-significant relationships among gloss and the antioxidant capacity of the skin and liver, acrolein levels in the liver, and uric acid levels in the liver indicate that guanine, and subsequently uric acid, may come from other sources besides guanine crystals contained in iridophores. Thus, our results cannot exclude the possibility that the decrease in gloss we observed was caused by UV-B radiation directly disrupting the molecular structure of the guanine crystals rather than depending on an oxidative pathway. The condition dependence of structural coloration may be explained by trade-offs in guanine usage between coloration and
Table 1. Uric acid in the livers of convict cichlids Amatitlania nigrofasciata is associated with greater antioxidant potential (Trolox equivalent antioxidant capacity) in the liver (overall model R2 < 0.16, F3,38 ¼ 2.45, P ¼ 0.08).
Intercept Uric acid Body condition Uric acid * body condition
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Estimate
Standard error
t-value
P-value
6.70e01 9.63e01 8.91e þ 03 2.61e þ 04
1.66e01 4.23e01 4.24e þ 03 1.06e þ 04
4.04 2.28 2.10 2.45
0.003 0.03 0.04 0.02
STRUCTURAL COLORATION AND OXIDATIVE STRESS conversion to uric acid. Another likely explanation, termed the shared-pathway hypothesis, is that essential pathways may be common to both vital physiological processes and ornament production or maintenance (Hill, 2011). For example, Hill (2014) has proposed that signal quality may be affected by the efficiency of cellular respiration, a process that could affect immune function, oxidative balance, as well as the synthetic pathways necessary for the production or maintenance of structural coloration. If exposure to UV-B light or dietary intake of guanine alter the efficiency of cellular respiration (particularly oxidative phosphorylation; Hill, 2014), then iridophore expression and uric acid production could simultaneously be affected. Future studies of iridophore expression in this and other fish species should focus on the relationship between ornament production and other important cellular processes, rather than simply on resource trade-offs (Hill, 2011). Our gloss measurement, a modification of the gloss measurement used by Maia et al. (2011), has both shortcomings and advantages for widespread use. A potential limitation is that this method uses only one measure each for the diffuse (25°) and the spectral (45°) reflectance. More accurate measurements could be made using an integrating sphere or glossometer. However, our method can be easily and inexpensively employed. Furthermore, it can be used on live specimens (as we did here), allowing researchers to track changes in structural coloration of different body regions throughout the course of an experiment. It also lends itself well to comparative studies, which is particularly advantageous because gloss may represent an evolutionary transition from matte black to iridescence (Toomey et al., 2010; Maia et al., 2011). In addition to studies of comparative coloration or shared physiological pathways, future research on gloss should focus upon interactions between structural coloration and pigmentation (San-Jose et al., 2013). For example, how does iridophore expression affect the quality of camouflage or the expression of pigment-based signals? This question was recently addressed by Wucherer and Michiels (2014), who elucidated a role of iridophores in the expression of red fluorescence in the iris of the cryptic marine fish Tripterygion delaisi. Theoretical approaches have begun to classify how structural coloration can affect carotenoid signals; the ability to rapidly and inexpensively measure gloss could help create an in vivo approach (Grether et al., 2004). In the design of our experiment, we purposefully chose a location on the fish lacking in pigment, both melanin and carotenoids, in order to limit background noise in assessing gloss. Future research should focus on the complex interactions among iridophores and other classes of chromatophores in order to improve our understanding of structural and pigment-based coloration.
CONCLUSIONS In our study, we showed that iridophore-based coloration is influenced by nutritional state in female convict cichlids Amatitlania nigrofasciata. We found that expression of structural
365 coloration was positively correlated with guanine in the skin, a relationship we determined using Hunter's contrast gloss (a simple, non-invasive method for measuring the ratio of specular to diffuse reflectance) and high performance liquid chromatography. Exposure to UV-B light resulted in a decrease in gloss, but did not induce oxidative stress in a way that we could detect. Our work suggests that structural coloration may be conditiondependent in fish, but future research should focus on metabolic pathways that are common to both iridophore expression and antioxidant processes.
ACKNOWLEDGMENTS Renae Brodie, Geoff Hill, Beth Jakob, and an anonymous reviewer made helpful comments on earlier versions of this manuscript. Financial support was provided by the Dayton Fund at Amherst College, the Department of Biology at Amherst College, and a grant from the National Science Foundation (IOS-1051598) to E.D.C.
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STRUCTURAL COLORATION AND OXIDATIVE STRESS Raghavan S, Kristinsson HG, Leeuwenburgh C. 2008. Radical scavenging and reducing ability of tilapia (Oreochromis niloticus) protein hydrolysates. J Agric Food Chem 56:10359–10367. Ramadan A, Afifi NA, Moustafa MM, Sarny AM. 1994. The effect of ascogen on the immune response of Tilapia fish to Aeromonas hydrophilia vaccine. Fish Shellfish Immun 4:159–165. Rasmussen PV, Dyck J. 2000. Silkiness in brown mink pelts characterized with optical methods. J Anim Sci 78:1967–1709. Robinson ML, Gornez-Raya L, Rauw WM, Peacock MM. 2008. Fulton's body condition factor K correlates with survival time in a thermal challenge experiment in juvenile Lahontan cutthroat trout (Oncorhynchus clarki henshawi). J Thermal Biol 33:363–368. Rohrlich ST, Rubin RW. 1975. Biochemical characterization of crystals from the dermal iridophores of a chameleon Anolis carolinensis. J Cell Biol 66:635–645. Salo HM, Jokinen EI, Markkula SE, Aaltonen TM, Penttila HT. 2000. Comparative effects of UVA and UVB irradiation on the immune system of fish. J Photochem Photobiol B 56:154–162. San-Jose LM, Granado-Lorencio F, Sinervo B, Fitze P. 2013. Iridophores and not carotenoids account for chromatic variation of carotenoidbased coloration in common lizards (Lacerta vivipara). Am Nat 181:396–409. Sautin YY, Johnson RJ. 2008. Uric acid: the oxidant-antioxidant paradox. Nucleos Nucleot Nucl 27:608–619. Saranathan V, Osuji CO, Mochrie SGJ, et al. 2010. Structure, function, and self-assembly of single network gyroid (l4132) photonic
367 crystals in butterfly wing scales. Proc Natl Acad Sci USA 107:11676–11681. Siefferman L, Hill GE. 2005. Evidence for sexual selection on structural plumage coloration in female eastern bluebirds (Sialia sialis). Evolution 59:1819–1828. Taylor JD. 1969. The effects of intermedin on the ultrastructure of amphibian iridophores. Gen Comp Endocrinol 12:405–416. Toomey MB, Butler MW, Meadows MG, et al. 2010. A novel method for quantifying the glossiness of animals. Behav Ecol Sociobiol 64:1047–1055. Uchida K, Kanematsu M, Sakai K, et al. 1998. Protein-bound acrolein: potential markers for oxidative stress. Proc Natl Acad Sci USA 95:4882–4887. von Schantz T, Bensch S, Grahn M, Hasselquist D, Wittzell H. 1999. Good genes, oxidative stress and condition-dependent sexual signals. Proc R Soc Lond B 266:1–12. Wisenden BD. 1994. Factors affecting reproductive success in freeranging convict cichlids Cichlasoma nigrofasciatum. Can J Zool 72:2177–2185. Wisenden BD. 1995. Reproductive behaviour of free-ranging convict cichlids, Cichlasoma nigrofasciatum. Environ Biol Fishes 43:121–134. Wucherer MF, Michiels NK. 2014. Regulation of red fluorescent light emission in a cryptic marine fish. Front Zool 11:1. Xue C, Yu G, Hirata T, Sakaguchi M, Terao J. 1998. Antioxidative activity of carp blood plasma on lipid peroxidation. Biosci Biotechnol Biochem 62:201–205.
J. Exp. Zool.
Guanine-Based Structural Coloration as an Indicator of Oxidative Stress in a Cichlid Fish MATTHEW D. CAHN1, ALEXANDRIA C. BROWN1,2, 1 AND ETHAN D. CLOTFELTER * 1
Department of Biology, Amherst College, Amherst, Massachusetts Graduate Program in Organismic and Evolutionary Biology, University of Massachusetts, Amherst, Massachusetts 2
ABSTRACT
J. Exp. Zool. 323A:359–367, 2015
Vertebrate pigmentation is known to be influenced by oxidative stress, but few studies have tested the hypothesis that structural coloration can be similarly affected. We tested whether fish iridophores, which produce structural color using guanine stacks, might be affected by the prooxidant–antioxidant balance of the animal. Specifically, we hypothesized that convict cichlids (Amatitlania nigrofasciata) metabolize guanine present in iridophores to uric acid, an antioxidant, in response to oxidative damage. We used Hunter's contrast gloss and high performance liquid chromatography to determine whether dietary guanine supplementation allows fish to maintain their structural coloration despite oxidative stress induced via ultraviolet-B (UV-B) radiation. We found that dietary guanine was associated with greater skin gloss, and that exposure to UV-B light reduced glossiness. UV-B exposure did not increase oxidative damage (acrolein) or total antioxidant capacity in the skin or liver. Our experiment did not detect effects of dietary guanine or UV-B light on uric acid, but uric acid was positively related to antioxidant capacity. Our results support the hypothesis that structural color in fish may be altered by environmental stressors such as exposure to UV light, and highlight the need for future studies to consider the role of iridophores in condition-dependent visual signaling. J. Exp. Zool. 323A:359–367, 2015. © 2015 Wiley Periodicals, Inc. How to cite this article: Cahn MD, Brown AC, Clotfelter ED. 2015. Guanine-based structural coloration as an indicator of oxidative stress in a cichlid fish. J. Exp. Zool. 323A:359–367.
A large body of literature suggests that pigment-based signals can indicate the condition of the signaler in a variety of contexts: immune condition (Blount et al., 2003; McGraw and Ardia, 2003), nutritional condition (Hill and Montgomerie, '94), and oxidative stress (Pike et al., 2007). However, animal coloration can also be produced by structural elements. Structural coloration is an endogenous mechanism for producing color. In birds, structural coloration arises from combinations of keratin, melanin, and air (Doucet et al., 2006). The striking varieties of colors expressed on butterfly wings are also produced by structural color (Saranathan et al., 2010). In both taxa, previous studies suggest that structural coloration can act as an important signal underlying male and
Grant sponsor: National Science Foundation, Division of Integrative Organismal Systems; grant number: #1051598; grant sponsor: Dayton Fund at Amherst College, the Department of Biology at Amherst College. Alexandria C. Brown’s current address: Division of Biostatistics and Epidemiology, School of Public Health and Health Sciences, University of Massachusetts, Amherst, MA. Correspondence to: Ethan D. Clotfelter, Department of Biology, AC#2237, Amherst College, Amherst, MA 01002. E-mail: [email protected] Received 25 July 2014; Revised 30 January 2015; Accepted 2 February 2015 DOI: 10.1002/jez.1926 Published online 6 April 2015 in Wiley Online Library (wileyonlinelibrary.com).
© 2015 WILEY PERIODICALS, INC.
360 female mate preferences (e.g., nymphalid butterflies Hypolimnas bolina, Kemp, 2007; blue tits Cyanistes caeruleus, Hunt et al., '99; Eastern bluebirds Sialia sialis, Siefferman and Hill, 2005). In contrast to the species mentioned above, structural coloration in fish (Levy-Lior et al., 2010), cephalopods (M€athger et al., 2009), reptiles (Cooper and Greenberg, '92), spiders (LevyLior et al., 2010; Hsiung et al., 2014), and amphibians (Taylor, '69) consists of specialized cells containing alternating layers of cytoplasm and guanine platelets. It is thought that highly refractive guanine crystals and less refractive cytoplasm alternate in multilayer stacks within the iridophores to refract light, creating its optical characteristics from the interference of light reflected from the layer (Ide and Hama, '72; Rohrlich and Rubin, '75; Levy-Lior et al., 2010). In fishes, structural coloration is responsible for the metallic luster, which may provide camouflage from multiple angles due to light scattering produced by these guanine platelets (McKenzie et al., '95) and may play a role in the production of some color patterns (e.g., zebrafish Danio rerio, Hirata et al., 2003). Although guanine can be synthesized endogenously, guanine-based iridophore expression has been shown to depend upon condition in lizards (Brown et al., '48; San-Jose et al., 2013). Furthermore, studies on economically important fishes have shown that dietary supplementation with guanine can enhance immunity and growth (Ramadan et al., '94; Li et al., 2004a,b). These results suggest that guanine-based structural coloration may be costly for the organism to create or maintain. The current study tests whether the light-scattering ability of iridophores is linked to physiological condition, specifically the prooxidant–antioxidant balance. Free radicals, also known as reactive oxidative metabolites (ROMs), are a family of highly reactive molecules that can damage intracellular components (Dowling and Simmons, 2009). Elevated levels of free radicals have been linked to reduced expression of other conditiondependent traits, such as pigment-based color (von Schantz et al., 1999), and may play a role in aging (Balaban et al., 2005), immune function (Chew and Park, 2004), and life history traits (Blount et al., 2003; Pike et al., 2007). When an organism cannot counter the effects of free-radical production, oxidative stress occurs (Monaghan et al., 2009). Uric acid is an efficient scavenger of singlet oxygen, hydroxyl radicals, and peroxyl radicals (Sautin and Johnson, 2008), and has been shown previously to act as an antioxidant in fishes (Xue et al., '98; Ciereszko et al., '99). Oxidative stress may affect iridophore-based structural coloration via a physiological tradeoff: guanine allocation to iridophores could decrease circulating levels of uric acid, a metabolite of guanine and an antioxidant, available for mediating detrimental effects of free radicals. Condition dependence could be enforced by the energetic or opportunity cost of synthesizing guanine molecules and assembling the molecules in the precise manner necessary for J. Exp. Zool.
CAHN ET AL. iridophore function, as suggested previously by other researchers (Li and Gatlin, 2006; Levy-Lior et al., 2008). In our study, we used Hunter's contrast gloss to quantify iridophore expression in a fish. Hunter's contrast gloss has been used to measure luster in various industrial applications (Nickerson, '57; O’Brien et al., 1984). More recently, Hunter's contrast gloss has been used to quantify the luster of birds’ feathers (Maia et al., 2011) and eggshells (Igic et al., 2015). This measure takes advantage of the physical properties of gloss or luster; if a larger proportion of light is reflected at an angle equal to the incident angle (specular reflectance) relative to light reflected at random angles (diffuse reflectance), the surface will appear glossier. Hunter's contrast gloss can be calculated as the ratio of specular to diffuse reflectance, as shown below: Hunter’s Contrast Gloss ¼
700nm
S
l¼350nm
Specular ReflectanceðlÞ Diffuse ReflectanceðlÞ
We validated Hunter's contrast gloss as a measure of guanine in fish skin using high performance liquid chromatography (HPLC). We hypothesized that elevated free radical levels would increase guanine conversion to the antioxidant uric acid, resulting in reduced structural color. Dietary guanine supplementation may prevent this reduction in structural coloration if guanine is limited by access to the carbon and nitrogen building blocks needed to assemble it. We tested these predictions using the convict cichlid (Amatitlania nigrofasciata). Convict cichlids are often used in behavioral studies (Wisenden, '95; Beeching et al., '98) and females possess ornamental coloration that males lack (Wisenden, '94). Several studies have investigated the pigmentation component of convict cichlid coloration (Beeching et al., 2002; Anderson et al., 2014; Brown et al., 2014), but, to date, the concept of gloss has not been used in this or any other species of fish to quantify structural coloration. To test the hypothesis that iridophore-based expression can be altered by oxidative stress in convict cichlids, a 2 2 experiment design was used in which fish were fed a diet supplemented with guanine or a non-supplemented control diet. Across these diet groups, fish were then exposed to ultraviolet-B (UV-B) light at a level shown previously to induce a stress response in fish (Salo et al., 2000), or to control lighting conditions. Spectral reflectance of the skin, quantities of uric acid and guanine in the skin, and oxidative stress parameters were measured to determine the effects of diet and UV-B light on gloss. If increased free radical production reduces gloss, this result will support the hypothesis that iridophore appearance may be related to condition. If this reduction in gloss is accompanied by an increase in uric acid in the skin, fish may be metabolizing the guanine in their iridophores to combat oxidative stress. Finally, if these negative effects can be rescued by the addition of guanine to the diet, iridophore-based color expression may also contain information about a signaler's
STRUCTURAL COLORATION AND OXIDATIVE STRESS environment and ability to absorb, transport, and build necessary molecules.
MATERIALS AND METHODS Study Animals and Experimental Diets Sexually mature female convict cichlids (A. nigrofasciata), which we identified by the presence of orange ventral coloration, were obtained from a commercial distributor. Fish acclimated to the laboratory for 2 days prior to being housed in individual 7-L tanks at 26 °C. Water was aerated with air stones and changed (50–75%) twice weekly. All handling and treatment of animals was approved by the Institutional Animal Care and Use Committee (IACUC) of Amherst College. The diet was a modified H440 diet with a low-nutrient base of cellulose, dextrin, and agar (Ellis et al., 2000; Brown et al., 2014). Pure guanine (no. 190691 MP Biomedicals, Santa Ana, CA, USA) was added to the supplemented diet at a concentration of 4 mg g1, a concentration consistent with other dietary nucleotide supplementation studies (Ramadan et al., '94; Li et al., 2004a,b). Fish were fed to satiation once daily with the guanine-supplemented diet or the nonsupplemented diet for 5 weeks prior to the oxidative challenge. Ultraviolet Light Exposure After 5 weeks on the experimental diets, half of the fish from each diet treatment group were exposed to UV-B radiation, while the other half was exposed to control lighting conditions. Fish were removed from their home tanks and placed in identical 7-L tanks placed 20 cm under a pair of Philips TL 40 W/12 RS light bulbs, which each emitted 40 W of UV-B light from 290–315 nm (Philips, Amsterdam, Netherlands). The UV-B light exposure lasted for 120 min, a dosage previously found to be non-lethal yet elicit stress responses in another fish, the common roach Rutilus rutilus (Salo et al., 2000). After irradiation, fish were returned to their original tanks and allowed to recover for 24 hr. Control fish were placed in the same tanks as the UV-B exposed fish for 120 min, but with the UV-B bulbs turned off.
361 Morphological and Color Measurements Fish were anesthetized 24 hr after UV-B exposure with a 2 g L1 tricaine methanesulfonate (Western Chemical Co., Ferndale, WA, USA) solution. Standard length (0.01 mm) and body mass (0.01 g) were measured prior to structural coloration quantification using spectrometry. Fish were placed on a damp sponge and spectrometric readings were taken 2 mm posterior from the operculum on each fish (Fig. 1A and B), a planar location typically lacking pigmentation. A deuterium-tungsten halogen lamp fiber-optic light source (Ocean Optics Jaz1, Dunedin, FL, USA) was mounted 5 mm above and 45° from the area to be measured. Opposite the light source, an Ocean Optics USB4000 spectrometer probe 5 mm from the fish collected and measured the light reflected at 45° and then at 25° (Fig. 1A). Reflectance spectra from the spectrometer were recorded using SpectraSuite software (version 2.0.159). Spectra were analyzed between 350 and 700 nm (Jackson, 2003; Brown et al., 2014). The spectrometer was set to use boxcar smoothing of five with three spectra averaged with an integration time of 100 ms. The ratio of specular reflectance (light captured by the detector at 45°) to diffuse reflectance (light captured by the detector at 25°) was used to quantify Hunter's contrast gloss (see equation above; Hunter, '37; Rasmussen and Dyck, 2000; Maia et al., 2011). We found that the amount of guanine in the skin (determined by high performance liquid chromatography, see below) significantly predicted the gloss measurement in the same region of the body (R2 ¼ 0.14, F1,35 ¼ 5.24, P ¼ 0.03; Fig. 2). Thus, we will hereafter consider gloss to be an indicator of the structural coloration in the skin. For all subsequent analyses involving gloss, the change in gloss from immediately prior to 1 day after UV-B exposure (or control) was used. Positive values indicate an increase, and negative values a decrease, in glossiness. After morphological and color measurements, fish were euthanized by cervical dislocation. Liver and skin samples were aseptically removed with surgical scissors and forceps and stored at 80 °C for later analysis.
Figure 1. Apparatus used to obtain Hunter's contrast gloss measurements from convict cichlids A. nigrofasciata, showing the location of the spectrometer probe and light source (A) and lateral view of a fish, showing the location of spectrometer measurements taken on live fish (B). Gloss measurements were taken 2 mm posterior of operculum, a location lacking melanin pigmentation. J. Exp. Zool.
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Figure 2. Guanine content in the skin (as measured by high performance liquid chromatography) and Hunter's contrast gloss of the skin of female A. nigrofasciata were positively correlated (R2 ¼ 0.14, F1,35 ¼ 5.24, P ¼ 0.03). The gloss measurement was sine transformed due to skew.
Uric Acid and Guanine Measurements To measure uric acid in the liver, livers were homogenized in 1 mL mg1 phosphate buffered saline and centrifuged. The uric acid content of the supernatant was determined by using a colorimetric uric acid assay kit (QuantiChrom no. DIUA-250, BioAssay, Hayward, CA, USA) prepared according to the manufacturer's instructions. Liver uric acid was quantified using a microplate reader (Bio-Tek, Winooski, VT, USA) at 590 nm. High performance liquid chromatography (HPLC) was used to quantify guanine and uric acid from the skin samples using a protocol adapted from Burdett et al. (2012). In brief, skin samples were homogenized in 20 volume (v:w) 0.52 mM sodium 1-pentanesulfonate (no. 76952 Sigma–Aldrich, St. Louis, MO, USA) and 0.20 M KH2PO4 (no. P5655 Sigma–Aldrich) monobasic at pH 3.5 mobile phase. Samples were centrifuged at 16,000g for 15 min and the supernatant was filtered through 0.22 mm Spin-X cellulose acetate filter tubes (Sigma–Aldrich) at 16,000g for 5 min. The filtrate was run on a Waters 600 HPLC (Waters Corp., Milford, MA, USA) using a C18 column. The flow rate was set at 1.0 mL min1 with an injection loop size of 12 mL. AWaters 600 E wavelength absorbance detector produced a chromatogram at 254 nm. Peaks were integrated and compared to a guanine standard by the Empower 3 HPLC software (Waters Corp.). Oxidative Stress Assays Two assays were used to measure oxidative stress parameters in the fish. The first, which measures acrolein, detects lipid J. Exp. Zool.
peroxidation (Uchida et al., '98; Kurtz et al., 2006). The second assay was a Trolox equivalent antioxidant capacity (TEAC) assay, which measures the ability of a biological sample to quench free radicals as compared to Trolox, a synthetic analog of vitamin E. For the acrolein assay, liver protein was adjusted to 1 mg mL1 using a Bradford protein assay (Bio-Rad, Hercules, CA, USA). As described previously (Kurtz et al., 2006; Brown et al., 2014), the acrolein assay was performed using the monoclonal antibody (mAb5F6, no. ADI-KAMCC101-E, Enzo Life Sciences, Farmingdale, NY, USA) and horseradish peroxidase antibody from mice as the secondary antibody (no. A9044 Sigma–Aldrich). SigmaFast OPD (no. P9187) was the probe that allowed for acrolein quantification at 474 nm. Standards were compared to a standard curve of acrolein-modified bovine serum. Amounts of acrolein are expressed as mM acrolein mg1 total protein. The TEAC assay quantified the antioxidant capacity of fish livers and skin relative to Trolox (Amar et al., 2000; Raghavan et al., 2008). Liver and skin homogenates were prepared as for the acrolein assay. The assay uses 2,20 -azino(3-ethylbenzthiazoline-6-sulphonic acid) (no. A1888 Sigma–Aldrich), which has a blue color in its free-radical form and is colorless when quenched. Thus, lower TEAC values due to reduced absorbance in the spectrometer are indicative of greater antioxidant capacity. Serial dilutions of Trolox were used to construct the standard curve. The antioxidant capacities of Trolox and the
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homogenates were quantified at 734 nm using a microplate reader (Bio-Tek, Winooski, VT, USA). Statistical Analyses All statistical tests were performed using R version 3.0.1. Log transformations (liver TEAC) or sine transformations (liver acrolein, skin TEAC, and Hunter's contrast gloss) were applied to achieve normality of residuals. The effect of dietary guanine supplementation and UV-B exposure on structural coloration and oxidative stress parameters was compared using ANCOVA. Linear regression tested the relationship between structural coloration and oxidative stress parameters or guanine content of the skin, with body condition as a covariate. Linear regression also tested the effect of uric acid on oxidative stress. Fulton's body condition (Bolger and Connolly, '89; Meka and McCormick, 2005; Robinson et al., 2008) was calculated using the following equation: body mass (standard length3)1, and was included in analyses where it was significant. Differences were considered statistically significant when P < 0.05.
RESULTS Data were collected from 66 female A. nigrofasciata, which had a mean ( s.e.m.) body mass of 1.89 0.47 g and a mean standard length of 3.77 0.47 mm. Thirteen fish were assigned to the control diet þ no UV-B group, 14 fish were assigned to the control diet þ UV-B group, 15 fish were assigned to the guaninesupplemented diet þ no UV-B group, and 13 fish were assigned to the guanine-supplemented diet þ UV-B group. In analyses with n < 66, differences in sample sizes were due to fish tissues or organs too small to analyze. The reflectance spectra obtained from a representative fish are shown in Figure 3; these values were used to calculate our gloss measurement. We found no differences in skin gloss between convict cichlids maintained on the guanine diet compared to the control diet (F1,45 ¼ 0.80, P ¼ 0.38; Fig. 4), but as described above, the guanine content of a fish's skin was positively related to its gloss (R2 ¼ 0.14, F1,35 ¼ 5.24, P ¼ 0.03; Fig. 2). Skin gloss was significantly reduced by exposure to UV-B light (F1,45 ¼ 5.47, P ¼ 0.02; Fig. 4). The interaction between guanine diet and UV-B light was not significant (F1,45 ¼ 1.26, P ¼ 0.27). We quantified acrolein in the liver as an indicator of oxidative damage, and used the Trolox equivalent antioxidant capacity (TEAC) assay to measure the antioxidant capacity of the liver and the skin. To verify these measures, we regressed liver acrolein against liver TEAC and found (as predicted) a significant negative relationship (R2 ¼ 0.27, F1,40 ¼ 15.9, P ¼ 0.0003). When we exposed convict cichlids to UV-B light for 2 hr and collected tissue samples the following day, there were no significant changes in liver acrolein (F1,47 ¼1.55, P ¼ 0.22), liver TEAC (F1,38 ¼ 0.80, P ¼ 0.38), or skin TEAC (F1,46 ¼ 0.03, P ¼ 0.87). Furthermore, exposure to UV-B light did not appear to affect
Figure 3. Reflectance spectra (from 350–700 nm) of a representative A. nigrofasciata. The solid line shows the specular reflectance with both the spectrometer probe and light source at 45°. The dashed line shows the diffuse reflectance with the light source at 45° and the spectrometer probe at 25°. These values were used to calculate our gloss measurement (see Materials and Methods).
the conversion of guanine to uric acid, as uric acid levels were similar between the UV-B and control light groups (F1,47 ¼ 0.01, P ¼ 0.92). Fish fed the diet supplemented with guanine showed no significant differences in liver acrolein (F1,47 ¼ 0.11, P ¼ 0.74), liver TEAC (F1,38 ¼ 0.18, P ¼ 0.67), or TEAC in the skin (F1,46 ¼ 0.02, P ¼ 0.88). There were no significant differences in the amount of uric acid in the liver between fish on the guanine diet and fish on the control diet (F1,47 ¼ 0.53, P ¼ 0.47). The change in skin gloss during the experiment was not a significant predictor of acrolein in the liver (R2 ¼ 0.004, F1,43 ¼ 0.16, P ¼ 0.69), the antioxidant capacity (TEAC) of the liver (R2 ¼ 0.005, F1,39 ¼ 0.19, P ¼ 0.67), or the antioxidant capacity of the skin (R2 ¼ 0.006, F1,42 ¼ 0.24, P ¼ 0.63). Furthermore, the change in glossiness did not predict the amount of uric acid present in the liver (R2 ¼ 0.017, F1,43 ¼ 0.78, P ¼ 0.38). Uric acid was not a significant predictor of acrolein in the liver (R2 < 0.001, F1,49 < 0.001, P ¼ 0.99), but it did explain variation in antioxidant capacity of the liver (P ¼ 0.03; Table 1). Uric acid was not a significant predictor of the antioxidant capacity of the skin (R2 < 0.08, F3,46 ¼ 1.35, P ¼ 0.27). J. Exp. Zool.
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Figure 4. Change in skin gloss following UV-B exposure in female A. nigrofasciata (mean s.e.m.) maintained on a control diet or a guanine-supplemented diet. The symbols represent fish exposed to UV-B light (triangles) and control light conditions (circles). The gloss measurement was sine transformed due to skew. UV-B exposure significantly reduced skin gloss (F1,45 ¼ 5.47, P ¼ 0.02). Neither diet (F1,45 ¼ 0.80, P ¼ 0.38) nor the diet UV interaction (F1,45 ¼ 1.26, P ¼ 0.27) had an effect on gloss.
DISCUSSION In this study, we tested the hypothesis that structural coloration in female convict cichlids A. nigrofasciata is an indicator of physiological condition. We used gloss as a proxy for structural color and then measured changes in gloss in fish fed a diet supplemented with guanine (precursor to the antioxidant uric acid) and in fish in which we disrupted the prooxidant– antioxidant balance via exposure to UV-B light. The first prediction was that glossier skin is associated with greater levels of guanine in the skin, a prediction for which we found some support. We used high performance liquid chromatography (HPLC) to quantify the guanine in fish skin, and found that it was positively related to the gloss measurement taken at that same location when all fish in the experiment were pooled together. However, when we experimentally manipulated fish
diets, we found no differences in skin gloss between diet treatment groups. This absence of an effect may be due to several factors: modest sample sizes; insufficient duration of the diet period (Brown et al., 2014); individual variation in food intake rates (Martins et al., 2011); individual variation in the rates at which guanine is assimilated and deposited in the skin; and competing needs for guanine for other physiological functions. However, it is also possible, contrary to our hypothesis, that dietary guanine supplementation does not affect guanine deposition or skin gloss. Our second prediction was that structural coloration in female convict cichlids is an indicator of oxidative stress. Again, our findings provide mixed support for this prediction. Exposure to UV-B light caused a significant reduction in skin gloss in our experiment. However, UV-B exposure caused no measurable increases in liver acrolein, a lipid peroxidation product widely used as biomarker for oxidative stress (Uchida et al. '98), nor did it decrease antioxidant capacity in either the liver or the skin. Previous studies have shown that UV radiation causes oxidative stress in marine environments (Palenik et al., '91; Dahms and Lee, 2010). Therefore, our lack of effect could be caused by an inability to detect a change in oxidative stress, perhaps due to insufficient UV-B exposure. Nevertheless, UV radiation has also been shown to alter DNA directly, depress immune function, and induce apoptosis in fishes (Ahmed and Setlow, '93; Blazer et al., '97; Salo et al., 2000). Therefore, if UV-B exposure did not induce detectable levels of oxidative stress, differences between UV-B exposed and control groups in terms of iridophore expression can be attributed to its role as a stressor of various physiologically relevant activities. The non-significant relationships among gloss and the antioxidant capacity of the skin and liver, acrolein levels in the liver, and uric acid levels in the liver indicate that guanine, and subsequently uric acid, may come from other sources besides guanine crystals contained in iridophores. Thus, our results cannot exclude the possibility that the decrease in gloss we observed was caused by UV-B radiation directly disrupting the molecular structure of the guanine crystals rather than depending on an oxidative pathway. The condition dependence of structural coloration may be explained by trade-offs in guanine usage between coloration and
Table 1. Uric acid in the livers of convict cichlids Amatitlania nigrofasciata is associated with greater antioxidant potential (Trolox equivalent antioxidant capacity) in the liver (overall model R2 < 0.16, F3,38 ¼ 2.45, P ¼ 0.08).
Intercept Uric acid Body condition Uric acid * body condition
J. Exp. Zool.
Estimate
Standard error
t-value
P-value
6.70e01 9.63e01 8.91e þ 03 2.61e þ 04
1.66e01 4.23e01 4.24e þ 03 1.06e þ 04
4.04 2.28 2.10 2.45
0.003 0.03 0.04 0.02
STRUCTURAL COLORATION AND OXIDATIVE STRESS conversion to uric acid. Another likely explanation, termed the shared-pathway hypothesis, is that essential pathways may be common to both vital physiological processes and ornament production or maintenance (Hill, 2011). For example, Hill (2014) has proposed that signal quality may be affected by the efficiency of cellular respiration, a process that could affect immune function, oxidative balance, as well as the synthetic pathways necessary for the production or maintenance of structural coloration. If exposure to UV-B light or dietary intake of guanine alter the efficiency of cellular respiration (particularly oxidative phosphorylation; Hill, 2014), then iridophore expression and uric acid production could simultaneously be affected. Future studies of iridophore expression in this and other fish species should focus on the relationship between ornament production and other important cellular processes, rather than simply on resource trade-offs (Hill, 2011). Our gloss measurement, a modification of the gloss measurement used by Maia et al. (2011), has both shortcomings and advantages for widespread use. A potential limitation is that this method uses only one measure each for the diffuse (25°) and the spectral (45°) reflectance. More accurate measurements could be made using an integrating sphere or glossometer. However, our method can be easily and inexpensively employed. Furthermore, it can be used on live specimens (as we did here), allowing researchers to track changes in structural coloration of different body regions throughout the course of an experiment. It also lends itself well to comparative studies, which is particularly advantageous because gloss may represent an evolutionary transition from matte black to iridescence (Toomey et al., 2010; Maia et al., 2011). In addition to studies of comparative coloration or shared physiological pathways, future research on gloss should focus upon interactions between structural coloration and pigmentation (San-Jose et al., 2013). For example, how does iridophore expression affect the quality of camouflage or the expression of pigment-based signals? This question was recently addressed by Wucherer and Michiels (2014), who elucidated a role of iridophores in the expression of red fluorescence in the iris of the cryptic marine fish Tripterygion delaisi. Theoretical approaches have begun to classify how structural coloration can affect carotenoid signals; the ability to rapidly and inexpensively measure gloss could help create an in vivo approach (Grether et al., 2004). In the design of our experiment, we purposefully chose a location on the fish lacking in pigment, both melanin and carotenoids, in order to limit background noise in assessing gloss. Future research should focus on the complex interactions among iridophores and other classes of chromatophores in order to improve our understanding of structural and pigment-based coloration.
CONCLUSIONS In our study, we showed that iridophore-based coloration is influenced by nutritional state in female convict cichlids Amatitlania nigrofasciata. We found that expression of structural
365 coloration was positively correlated with guanine in the skin, a relationship we determined using Hunter's contrast gloss (a simple, non-invasive method for measuring the ratio of specular to diffuse reflectance) and high performance liquid chromatography. Exposure to UV-B light resulted in a decrease in gloss, but did not induce oxidative stress in a way that we could detect. Our work suggests that structural coloration may be conditiondependent in fish, but future research should focus on metabolic pathways that are common to both iridophore expression and antioxidant processes.
ACKNOWLEDGMENTS Renae Brodie, Geoff Hill, Beth Jakob, and an anonymous reviewer made helpful comments on earlier versions of this manuscript. Financial support was provided by the Dayton Fund at Amherst College, the Department of Biology at Amherst College, and a grant from the National Science Foundation (IOS-1051598) to E.D.C.
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